Mechanistic study of thermally denatured β-lactoglobulin nanoparticles for ferulic acid encapsulation and antioxidant enhancement

Ferulic acid (FA) is a potent natural antioxidant, but its poor solubility and stability limit its application. Here, heat-treated β-lactoglobulin (β-Lg) nanoparticles were developed for FA encapsulation. ITC confirmed 1:1 binding between β-Lg and FA, with a K d of 1.28 μM, driven mainly by hydrogen bonding and van der Waals interactions. Heating β-Lg and FA at 80 °C for 15 min yielded 31–34 nm nanoparticles with 61.61% encapsulation efficiency and favorable colloidal stability at a 1:1 M ratio. The nanoparticles exhibited enhanced radical-scavenging activity, with ABTS and DPPH IC₅₀ values of 0.18 and 0.44 mg/mL, respectively, and improved cellular antioxidant activity. Network pharmacology and ARE-luciferase assays indicated ARE-dependent transcriptional activation, consistent with Nrf2–ARE involvement. Molecular docking and molecular dynamics simulations revealed FA binding within the β-Lg hydrophobic calyx, anchored by PRO38. These findings demonstrate the potential of thermally denatured β-Lg as a nanocarrier for enhancing FA antioxidant activity.